A liquid chromatography is used for a separation or a detection of various materials, and especially for a separation or a detection of a hydrophilic material, for example, for a separation of protein from a biological sample, an ion exchange chromatography is used. Ion exchange chromatography is a method in which a carrier having an ion exchange group is utilized for separating materials depending upon a difference in the strength of an ionic bond of the material to be separated to the carrier. A weak cation exchange chromatography utilizing a carrier having a carboxyl group as the ion exchange group is effective for an analysis of protein or peptide.
For example, glycosylated hemoglobins in blood are measured with ion exchange chromatography utilizing such a carrier.
The glycosylated hemoglobins are formed by a non-enzymatical reaction of hemoglobin in an erythrocyte with glucose in the blood. Since a measurement of glycosylated hemoglobins reveals an average concentration of glucose in the blood, the measurement of the glycosylated hemoglobins is widely used for the diagnosis of diabetes. The glycosylated hemoglobins are now determined by a high performance liquid chromatography (hereinafter referred to as the "HPLC") mainly utilizing the above-mentioned carrier. The HPLC provides rapid measurement as compared with conventional column chromatography, electrophoresis, colorimetric analysis and the like.
An organic polymer carrier or an inorganic carrier is generally used as a carrier for weak cation exchange chromatography. A carrier gel of organic polymers used most frequently is a carrier in which carboxyl groups are introduced on a surface of a particle of a styrene-divinylbenzene crosslinked copolymer. Such a carrier is obtained by introducing carboxyl groups to a particle of a styrene-divinylbenzene crosslinked copolymer by a chemical reaction. For example, alkyl halide is introduced to a benzene ring of styrene in the copolymer by treating the particle of the copolymer with chloromethyl ether and the like, and the resultant material is hydrolyzed and oxidized, thereby introducing the carboxyl groups.
An example of the organic polymer carrier, besides the above-mentioned gel, includes particles of a crosslinked copolymer of styrene, divinylbenzene and a monomer having a carboxyl group. Moreover, Japanese Laid-Open Patent Publication No. 58-221164 discloses a carrier made of a copolymer of an acrylate or a methacrylate such as tetramethylolmethane triacrylate and acrylic acid or methacrylic acid. Such a carrier is generally prepared in a method disclosed in the foregoing Japanese Laid-Open Patent Publication No. 58-221164: for example, the crosslinked copolymer particles are prepared by adding a polymerization initiator to a mixture of a crosslinkable monomer and a monomer having a carboxyl group for a suspension polymerization. Alternatively, particles obtained by copolymerizing styrene, divinylbenzene and a monomer having a functional group which can be converted into a carboxyl group by a hydrolytic reaction (hereinafter referred to as the "hydrolyzable group") and then converting the functional group into a carboxyl group by a hydrolytic reaction can be used. It is necessary to increase the degree of the crosslink in order to improve the pressure resistance of such a carrier. However, when the degree of the crosslink is increased, the hydrophobicity of the gel is also increased since a crosslinked portion is hydrophobic, resulting in a nonspecific adsorption of protein. Therefore, the amount of a crosslinking agent to be used is limited, and it is difficult to obtain a satisfactory pressure resistance. Moreover, since the carrier obtained in the above-mentioned manner contains carboxyl groups in the entire copolymer particle, the carrier is likely to swell or shrink in an aqueous solvent. This is another reason for its insufficient pressure resistance.
The above-mentioned carrier used in determining glycosylated hemoglobins in high performance liquid chromatography shows the following defects:
Generally, glycosylated hemoglobins are determined by utilizing an eluent with a low eluting ability (hereinafter called "first eluent") and an eluent with a high eluting ability (hereinafter called "second eluent") in steps or in a linear gradient method. The first eluent increases a number of free carboxyl groups in the carrier particles. Thus, hemoglobin in a sample except for glycosylated hemoglobins is retained by the carrier, thereby separating and eluting the glycosylated hemoglobins. Since the second eluent has a large ionic strength, the free carboxyl groups become salts. Therefore, the retained hemoglobin except for the glycosylated hemoglobins is rapidly eluted.
However, in the above-mentioned carrier, especially in the carrier prepared by using a hydrophobic monomer and a hydrophilic monomer such as a monomer having a carboxyl group or a latent carboxyl group, ion exchange groups derived mainly from the hydrophilic monomer are present throughout the carrier particle. When such a carrier comes in contact with the second eluent, the carrier swells, thereby increasing pressure in the column. It is necessary to return the carboxyl groups to be free by letting the first eluent flow after measuring one sample in order to measure another sample. At this point, in order to return the ion exchange groups existing in the carrier to be sufficiently free, it is necessary to let a considerable amount of the first eluent flow. Therefore, a longer time is required for the measurement. Glycosylated hemoglobins can now be measured relatively rapidly by the HPLC utilizing a carrier gel of polymer. However, when attempting to increase the measuring rate, the separating ability is degraded, because the ion exchange groups in the carrier can not be sufficiently exchanged or the carrier swells. In order to achieve an accurate separation, it is necessary to decrease the eluting rate.
On the other hand, as an inorganic carrier used for the separation of protein from an ordinary biological sample, Japanese Laid-Open Patent Publication No. 63-75558 discloses a silica carrier in which carboxyl groups are chemically bonded to a surface of a porous silica gel. This carrier has a satisfactory pressure resistance and separating ability, and can provide a relatively rapid treatment. However, this gel has a characteristic of adsorbing a material having basic groups such as protein due to an effect of residual silanol groups on the surface thereof. Moreover, since silica gel is dissolved in an acid and an alkali, pH of the eluent is limited from 3 to 8.
Moreover, a so called seed polymerization is disclosed in Japanese Laid-Open Patent Publication Nos. 56-151712, 59-18705, 62-63856 and 63-79064 as a method for obtaining a carrier which can be used as a carrier in weak cation exchange chromatography and has a relatively satisfactory pressure resistance. This method is such that crosslinked polymer particles are impregnated with a polymerization initiator and a monomer, and the impregnated particles are suspension-polymerized in order to obtain two-layered particles. When a monomer having a carboxyl group is used as the monomer for impregnating the crosslinked polymer particles in this method, a carrier for weak cation exchange chromatography can be obtained. A similar carrier for weak cation exchange chromatography as the above can be obtained by impregnating the particles with a monomer having a functional group which can be converted into a hydrolyzable group, polymerizing the said monomer, and then hydrolyzing the hydrolyzable groups owned by the particles. However, since the thus obtained particles have carboxyl groups therein, the particles are likely to swell or shrink in an aqueous solvent due to the foregoing reasons, resulting in an insufficient pressure resistance. Moreover, there are defects in that the separating ability of liquid chromatography is degraded or it takes a longer time for the separation when such a carrier is used.